1. Field of the Invention
The invention relates to a process for the fractionation of animal blood plasma and the isolation and purification of natural, unaltered, undenatured immune gamma globulin and albumin. More particularly, the invention relates to the preparation of intravenously injectable human immune gamma globulin.
The plasma fractionation industry in this country and Europe currently uses the method of cold ethanol fractionation developed by Dr. E. J. Cohn of Harvard University in the 1940's. Even though new methods of protein separation have evolved in the ensuing 35 years, the industry has neither chosen to gamble on new methods nor face up to any regulatory inhibition that a new product faces. As a result of this lack of new spirit, no methods have been developed to produce a new large scale system that would lead to a cheaper and better product. It has been demonstrated that there are severe limitations to the Cohn method, the major being that the process does indeed denature some of the proteins which it attempts to purify and isolate.
Several years have been spent in the development of methods and technology for the preparation and purification of animal (horse, goat and rabbit plasma with antibodies against human lymphocytes) plasma. This anti-lymphocyte globulin, a biologic immunosuppressive agent termed ALG, has been demonstrated to have potent immunosuppressive activity against cell mediated immunity, the type of immunity that causes grafts to be rejected.
After demonstrating the efficacy of this material in animals, it was sought to bring this to the clinic as an adjunctive immunosuppressive agent in the management of the human renal transplant patients. At the time of clinical application of this material, it was felt that the following criteria had to be established: safety of the preparation, a method for fractionating and isolating maximal activity in plasma and a method of fractionation that would allow administration of this material to patients by the most effective route. Animal experiments had demonstrated that the administration of the same quantity of IgG by the intravenous route was two to three fold more effective than the intramuscular or other route. In addition, only limited quantities can be administered intramuscularly whereas 10 to 100 times as much can be administered intravenously. Therefore it was sought to produce a preparation of horse anti-lymphocyte globulin that could be administered safely by the intravenous route. This was done with full knowledge that human gamma globulin could not be administered by the intravenous route.
Early studies by Good at Minnesota, Janeway at Harvard, and others, had demonstrated that the intravenous administration of the Cohn fractionated human gamma globulin often produced severe systemic reactions. The cause of these reactions was related to the high concentration of aggregated material due to the presence of a protein denaturing agent found in the fraction removed by the present invention. As a result, the only approved method for fractionation is the Cohn cold ethanol process and the only approved route of administration of gamma globulin today is the intra-muscular route.
Since intravenous administration of fresh plasma produces no reaction, it has been accepted that the aggregated materials were there as a result of the denaturation of the gamma globulin by the alcohol method and, therefore, alternative methods were sought for the purification and preparation of the horse globulin materials. That this has been successful, and that the goal has been achieved of preparing a safe globulin that can be administered intravenously in patients, is supported by the following facts. Up to the present, over 30,000 grams of purified horse anti-lymphocyte globulin have been prepared. This material has been administered intravenously to over 900 renal transplant patients. The total experience with intravenous administrations approaches over 13,000 separate administrations. These intravenous administrations have not been associated with any evidence of reaction at the time of administration. It therefore was felt that the methods utilized in the preparation and isolation of these horse, goat and rabbit gamma globulins could resolve the problems associated with the attempts to produce and prepare from human plasma an intravenous gamma globulin for use in treating life threatening viral and bacterial infections.
Since viral and bacterial infections constitute the leading cause of patient death in renal transplantation, methods were sought that could be used to treat these severe life threatening infections. One attractive approach was the use of a human gamma globulin that could be administered intravenously. Knowing that none of the commercial preparations could be used in this manner, the possibility was explored of fractionating human gamma globulin from plasma according to the methods developed for the fractionation of the animal proteins. It was found in general principle that this could be done. In clinical studies with this new IV human gamma globulin, doses have been administered intravenously ranging from 20 mg/kg/day to 200 mg/kg/day over a 14 day period. Studies to date show promise of establishing efficacy of the intravenous administration of gamma globulin isolated from pooled human plasma in the treatment of life threatening viral and bacterial infections in immunologically compromised patients.
2. Description of the Prior Art
Although there have been numerous attempts to produce an intravenously injectable gamma globulin, none to our knowledge has produced a natural, unaltered and undenatured product. These efforts are reviewed in Stephan (Biotest) U.S. Pat. No. 3,916,026 and Pappenhagen et al (Cutter) U.S. Pat. No. 3,903,262. The Stephan patent discloses the preparation of intravenously injectable non-complement binding gamma globulin by treating complement binding gamma globulin with beta-propiolactone. The Pappenhagen et al patent discloses preparing intravenously injectable modified immune serum globulin by cleaving at least one interchain disulfide linkage of intact immune serum globulin chains having intact intrachain disulfide linkages and replacing the cleaved disulfide linkage with a pair of alkylate mercapto groups.